Novel TNF receptor death domain ligand proteins and inhibitors of ligand binding

ABSTRACT

Novel TNF receptor death domain (“TNF-R1-DD”) ligand proteins are disclosed. Polynucleotides encoding the TNF-R1-DD ligand protein are also disclosed, along with vectors, host cells, and methods of making the TNF-R1-DD ligand protein. Pharmaceutical compositions containing the TNF-R1-DD ligand protein, methods of treating inflammatory conditions, and methods of inhibiting TNF-R death domain binding are also disclosed. Methods of identifying inhibitors of TNF-R death domain binding and inhibitors identified by such methods are also disclosed.

This application is a continuation-in-part of application Ser. No.08/602,228, filed Feb. 15, 1996, which was a continuation-in-part ofapplication Ser. No. 08/533,901, filed Sep. 26, 1995, which was acontinuation-in-part of application Ser. No. 08/494,440, filed Jun. 19,1995, which was a continuation-in-part of application Ser. No.08/327,514, filed Oct. 19, 1994.

BACKGROUND OF THE INVENTION

The present invention relates to the field of anti-inflammatorysubstances and other substances which act by inhibiting binding to theintracellular domain of a tumor necrosis factor receptor (hereinafter“TNF-R”), such as, for example, the P55 type (or TNF-R1) TNF receptor.More particularly, the present invention -is directed to novel ligandswhich bind to the TNF-R intracellular domain and to inhibition ormodulation of signal transduction by this receptor.

Tumor necrosis factor (herein “TNF”) is a cytokine which produces a widerange of cellular activities. TNF causes an inflammatory response, whichcan be beneficial, such as in mounting an immune response to a pathogen,or when overexpressed can lead to other detrimental effects ofinflammation.

The cellular effects of TNF are initiated by the binding of TNF to itsreceptors (TNF-Rs) on the surface of target cells. The isolation ofpolynucleotides encoding TNF-Rs and variant forms of such receptors hasbeen described in European patent publication Nos. EP 308,378, EP393,438, EP 433,900, EP 526.905 and EP 568,925; in PCT patentpublication Nos. WO91/03553 and WO93/19777: and by Schall et at., Cell61:361-370 (1990) (disclosing the P55 type TNF receptor). Processes forpurification of TNF-Rs have also been disclosed in U.S. Pat. No.5,296,592.

Native TNF-Rs are characterized by distinct extracellular transmembraneand intracellular domains. The primary purpose of the extracellulardomain is to present a binding site for TNF on the outside of the cell.When TNF is bound to the binding site, a “signal” is transmitted to theinside of the cell through the transmembrane and intracellular domains,indicating that binding has occurred. Transmission or “transduction” ofthe signal to the inside of the cell occurs by a change in conformationof the transmembrane and/or intracellular domains of the receptor. Thissignal is “received” by the binding of proteins and other molecules tothe intracellular domain of the receptor, resulting in the effects seenupon TNF stimulation. Two distinct TNF receptors of ˜55 kd (“TNF-R1”)and ˜75 kd (“TNF-R2”) have been identified. Numerous studies withanti-TNF receptor antibodies have demonstrated that TNF-R1 is thereceptor which signals the majority of the pleiotropic activities ofTNF. Recently, the domain required for signaling cytotoxicity and otherTNF-mediated responses has been mapped to the ˜80 amino acid near theC-terminus of TNF-R1. This domain is therefore termed the “death domain”(hereinafter referred to as “TNF-R death domain” and “TNF-R1-DD”) (see,Tartaglia et al., Cell 74:845-853 (1993)).

While TNF binding by TNF-Rs results in beneficial cellular effects, itis often desirable to prevent or deter TNF binding from causing otherdetrimental cellular effects. Although substantial effort has beenexpended investigating inhibition of TNF binding to the extracellulardomain of TNF-Rs, examination of binding of proteins and other moleculesto the intracellular domain of TNF-Rs has received much less attention.

However, ligands which bind to the TNF-R intracellular domain have yetto be identified. It would be desirable to identify and isolate suchligands to examine their effects upon TNF-R signal transduction andtheir use as therapeutic agents for treatment of TNF-induced conditions.Furthermore, identification of such ligands would provide a means forscreening for inhibitors of TNF-R/intracellular ligand binding, whichwill also be useful as anti-inflammatory agents.

SUMMARY OF THE INVENTION

Applicants have for the first time identified novel TNF-R1-DD ligandproteins and have isolated polynucleotides encoding such ligands.Applicants have also identified a known protein which may also bind tothe death domain of TNF-R.

In one embodiment, the present invention provides a compositioncomprising an isolated polynucleotide encoding a protein havingTNF-R1-DD ligand protein activity. In preferred embodiments, thepolynucleotide is selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO: 1from nucleotide 2 to nucleotide 1231;    -   (b) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:1;    -   (c) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:2;    -   (d) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:2;    -   (e) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:3 from nucleotide 2 to nucleotide 415;    -   (f) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:3;    -   (g) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:4;    -   (h) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ED NO:4;    -   (i) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:9 from nucleotide 2 to nucleotide 931;    -   (j) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ [D NO:9;    -   (k) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:10;    -   (l) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID        NO:10;    -   (m) a polynucleotide comprising the nucleotide sequence of SEQ        [ID NO:11 from nucleotide 2 to nucleotide 1822:    -   (n) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:11:    -   (o) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:12;    -   (p) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID        NO:12;    -   (q) a polynucleotide comprising the nucleotide sequence of SEQ D        NO:13 from nucleotide 3 to nucleotide 2846;    -   (r) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:13, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (s) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:14;    -   (t) a polynucleotide encoding an TNF-R1-DD Ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:14        and having TNF-R1-DD ligand protein activity;    -   (u) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:15 from nucleotide 326 to nucleotide 5092;    -   (v) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:15;    -   (w) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:16;    -   (x) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID        NO:16;    -   (y) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:17 from nucleotide 14 to nucleotide 2404;    -   (z) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:17;    -   (aa) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:18;    -   (bb) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID        NO:18; and    -   (cc) a polynucleotide capable of hybridizing under stringent        conditions to any one of the polynucleotides specified in        (a)-(cc).        In certain preferred embodiments, the polynucleotide is operably        linked to an expression control sequence. The invention also        provides a host cell, including bacterial, yeast, insect and        mammalian cells, transformed with such polynucleotide        compositions.

Processes are also provided for producing an TNF-R1-DD ligand protein,which comprises:

-   -   (a) growing a culture of the host cell transformed with such        polynucleotide compositions in a suitable culture medium; and    -   (b) purifying the TNF-R1-DD ligand protein from the culture. The        ligand protein produced according to such methods is also        provided by the present invention.

Compositions comprising a protein having TNF-R1-DD ligand proteinactivity are also disclosed. In preferred embodiments the proteincomprises an amino acid sequence selected from the group consisting of:

-   -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) fragments of the amino acid sequence of SEQ ID NO:2;    -   (c) the amino acid sequence of SEQ ID NO:4;    -   (d) fragments of the amino acid sequence of SEQ ID NO:4;    -   (e) the amino acid sequence of SEQ ID NO:6;    -   (f) fragments of the amino acid sequence of SEQ D) NO:6;    -   (g) the amino acid sequence of SEQ ID NO:10;    -   (h) fragments of the amino acid sequence of SEQ ID NO:10;    -   (i) the amino acid sequence of SEQ ID NO:12;    -   (j) fragments of the amino acid sequence of SEQ ID NO:12;    -   (k) the amino acid sequence of SEQ ID NO:14;    -   (l) fragments of the amino acid sequence of SEQ ID NO:14;    -   (m) the amino acid sequence of SEQ ID NO:16;    -   (n) fragments of the amino acid sequence of SEQ ID NO:16;    -   (o) the amino acid sequence of SEQ ID NO:18; and    -   (p) fragments of the amino acid sequence of SEQ ID NO:18:        the protein being substantially free from other mammalian        proteins. Such compositions may further comprise a        pharmaceutically acceptable carrier.

Compositions comprising an antibody which specifically reacts with suchTNF-R1-DD ligand protein are also provided by the present invention.

Methods are also provided for identifying an inhibitor of TNF-R deathdomain binding which comprise:

-   -   (a) combining an TNF-R death domain protein with an TNF-R1-DD        ligand protein, said combination forming a first binding        mixture;    -   (b) measuring the amount of binding between the TNF-R death        domain protein and the TNF-R1-DD ligand protein in the first        binding mixture;    -   (c) combining a compound with the TNF-R death domain protein and        an TNF-R1-DD ligand protein to form a second binding mixture;    -   (d) measuring the amount of binding in the second binding        mixture; and    -   (e) comparing the amount of binding in the first binding mixture        with the amount of binding in the second binding mixture;        wherein the compound is capable of inhibiting TNF-R death domain        binding when a decrease in the amount of binding of the second        binding mixture occurs. In certain preferred embodiments the        TNF-R1-DD ligand protein used in such method comprises an amino        acid sequence selected from the group consisting of:    -   (a) the amino acid sequence of SEQ ID NO:2;    -   (b) fragments of the amino acid sequence of SEQ ID NO:2;    -   (c) the amino acid sequence of SEQ ID NO:4;    -   (d) fragments of the amino acid sequence of SEQ ID NO:4;    -   (e) the amino acid sequence of SEQ ID NO:6;    -   (f) fragments of the amino acid sequence of SEQ ED NO:6;    -   (g) the amino acid sequence of SEQ ID NO:8;    -   (h) fragments of the amino acid sequence of SEQ ID NO:8    -   (i) the amino acid sequence of SEQ ID NO:10;    -   (j) fragments of the amino acid sequence of SEQ ID NO:10:    -   (k) the amino acid sequence of SEQ ID NO:12:    -   (l) fragments of the amino acid sequence of SEQ ID NO:12:    -   (m) the amino acid sequence of SEQ ID NO:14:    -   (n) fragments of the amino acid sequence of SEQ ID NO:14:    -   (o) the amino acid sequence of SEQ ID NO:16;    -   (p) fragments of the amino acid sequence of SEQ ID NO:16:    -   (q) the amino acid sequence of SEQ ID NO:18;    -   (r) fragments of the amino acid sequence of SEQ ID NO:18.        Compositions comprising inhibitors identified according to such        method are also provided. Such compositions may include        pharmaceutically acceptable carriers.

Methods are also provided for preventing or ameliorating an inflammatorycondition which comprises administering a therapeutically effectiveamount of a composition comprising a protein having TNF-R1-DD ligandprotein activity and a pharmaceutically acceptable carrier.

Other embodiments provide methods of inhibiting TNF-R death domainbinding comprising administering a therapeutically effective amount of acomposition comprising a protein having TNF-R1-DD ligand proteinactivity and a pharmaceutically acceptable carrier.

Methods are also provided for preventing or ameliorating an inflammatorycondition which comprises administering to a mammalian subject atherapeutically effective amount of a composition comprising apharmaceutically acceptable carrier and a protein selected from thegroup consisting of insulin-like growth factor binding protein-5(“IGFBP-5”), and fragments thereof having TNF-R1-DD ligand proteinactivity. Such proteins may also be administered for inhibiting TNF-Rdeath domain binding.

Methods of preventing or ameliorating an inflammatory condition or ofinhibiting TNF-R death domain binding are provided, which compriseadministering to a mammalian subject a therapeutically effective amountof inhibitors of TNF-R death domain binding, are also provided.

Methods of identifying an inhibitor of TNF-R death domain binding arealso provided by the present invention which comprise:

-   -   (a) transforming a cell with a first polynucleotide encoding an        TNF-R death domain protein, a second polynucleotide encoding an        TNF-R1-DD ligand protein, and at least one reporter gene,        wherein the expression of the reporter gene is regulated by the        binding of the TNF-R1-DD ligand protein encoded by the second        polynucleotide to the TNF-R death domain protein encoded by the        first polynucleotide;    -   (b) growing the cell in the presence of and in the absence of a        compound; and    -   (c) comparing the degree of expression of the reporter gene in        the presence of and in the absence of the compound;        wherein the compound is capable of inhibiting TNF-R death domain        binding when a decrease in the degree of expression of the        reporter gene occurs. In preferred embodiments, the cell is a        yeast cell and the second polynucleotide is selected from the        group consisting of:    -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:1 from nucleotide 2 to nucleotide 1231;    -   (b) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:1, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (c) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:2;    -   (d) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:2        and having TNF-R1-DD ligand protein activity;    -   (e) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:3 from nucleotide 2 to nucleotide 415;    -   (f) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:3, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (g) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:4;    -   (h) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:4        and having TNF-R1-DD ligand protein activity;    -   (i) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:5 from nucleotide 2 to nucleotide 559:    -   (j) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:5, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (k) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:6;    -   (l) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:6        and having TNF-R1-DD ligand protein activity;    -   (m) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:7 from nucleotide 57 to nucleotide 875;    -   (n) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:7, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (o) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:8;    -   (p) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:8        and having TNF-R1-DD ligand protein activity;    -   (q) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:9 from nucleotide 2 to nucleotide 931;    -   (r) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:9;    -   (s) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:10;    -   (t) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ED        NO:10;    -   (u) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:11 from nucleotide 2 to nucleotide 1822;    -   (v) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ D NO:11;    -   (w) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:12:    -   (x) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID        NO:12;    -   (y) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:13 from nucleotide 3 to nucleotide 2846;    -   (z) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:13, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (aa) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:14;    -   (bb) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:14        and having TNF-R1-DD ligand protein activity;    -   (cc) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:15 from nucleotide 326 to nucleotide 5092;    -   (dd) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:15, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (ee) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:16;    -   (ff) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ED NO:16        and having TNF-R1-DD ligand protein activity;    -   (gg) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO:17 from nucleotide 14 to nucleotide 2404;    -   (hh) a polynucleotide comprising a fragment of the nucleotide        sequence of SEQ ID NO:17, which encodes a protein having        TNF-R1-DD ligand protein activity;    -   (ii) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising the amino acid sequence of SEQ ID NO:18;    -   (jj) a polynucleotide encoding an TNF-R1-DD ligand protein        comprising a fragment of the amino acid sequence of SEQ ID NO:18        and having TNF-R1-DD ligand protein activity: and    -   (kk) a polynucleotide capable of hybridizing under stringent        conditions to any one of the polynucleotides specified in        (a)-(jj), which encodes a protein having TNF-R1-DD ligand        protein activity.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 depict autoradiographs demonstrating the expression ofTNF-R1-DD ligand proteins of the present invention.

FIG. 3 depicts an autoradiograph demonstrating the expression of clones1TU, 15TU and 27TU.

FIG. 4 demonstrates the binding of 1TU and 27TU to TNF-R1-DD. MBP,MBP-1TU or MBP-27TU (3 μg) was incubated with glutathione beadscontaining 3 μg of either GST or GST-TNF-R1-DD in 100 μl of bindingbuffer (0.2% Triton, 20 mM Tris pH 7.5, 140 mM NaCl, 0.1 mM EDTA, 10 mMDTT and 5% glycerol). The reaction ws performed at 4° C. for 2 hours andcentrifuged to remove unbound fraction (Unbound). The beads were thenwashed with 500 μl binding buffer four times and resuspended intoSDS-sample buffer (Bound). These samples were analyzed by Western blotusing anti-MBP antibody (New England Biolab).

FIG. 5 demonstrates the ability of 15TU and 27TU to activate the JNKpathway. COS cells were contransfected with HA-tagged JNK1 and clones15tu or 27TU. Cells were left untreated or treated for 15 min with 50ng/ml TNF, and HA-JNK1 was immunoprecipitated with anti-HA antibody. JNKactivity was measured in an in vitro kinase assay using GST-c-jun (aminoacids 1-79) as substrate, and reactions were electrophoresed onSDS-PAGE.

FIG. 6 is an autoradiograph of an SDS-PAGE gel of conditioned media fromCOS cells transfected with clone 3TW.

FIG. 7 is an autoradiograph which demonstrates that an antisenseoligonucleotide derived from the sequence of clone 3TW inhibitsTNF-induced cPLA₂ phosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have for the first time identified and isolatednovel polynucleotides which encode proteins which bind to the TNF-Rdeath domain. As used herein “TNF-R” includes all receptors for tumornecrosis factor. The P55 type TNF-R is the preferred receptor forpracticing the present invention.

The sequence of a polynucleotide encoding one such protein is set forthin SEQ ID NO:1 from nucleotides 2 to 1231. This polynucleotide has beenidentified as “clone 2DD” The amino acid sequence of the TNF-R1-DDligand protein encoded by clone 2DD is set forth in SEQ ID NO:2. It isbelieved that clone 2DD is a partial cDNA clone of a longer full lengthcoding sequence. However, as demonstrated herein the protein encoded byclone 2DD does bind the death domain of TNF-R (i.e., has “TNF-R1-DDligand protein activity” as defined herein). Clone 2DD was depositedwith the American Type Culture Collection on Oct. 13, 1994 and given theaccession number ATCC 69706.

The protein encoded by clone 2DD is 410 amino acids in length. Noidentical or closely related sequences were found using BLASTN/BLASTX orFASTA searches. Therefore, clone 2DD encodes a novel protein.

The sequence of a polynucleotide encoding one such protein is set forthin SEQ ID NO:3 from nucleotides 2 to 415. This polynucleotide has beenidentified as “clone 3DD”. The amino acid sequence of the TNF-R1-DDligand protein encoded by clone 3DD is set forth in SEQ ID NO:4. It isbelieved that clone 3DD is a partial cDNA clone of a longer full lengthcoding sequence. However, as demonstrated herein the protein encoded byclone 3DD does bind the death domain of TNF-R (i.e., has “TNF-R1-DDligand protein activity” as defined herein). Clone 3DD was depositedwith the American Type Culture Collection on Oct. 13, 1994 and given theaccession number ATCC 69705.

The protein encoded by clone 3DD is 138 amino acids. No identical orclosely related sequences were found using BLASTN/BLASTX or FASTAsearches. Therefore, clone 3DD encodes a novel protein.

A full-length clone corresponding to clone 3DD was also isolated andidentified as “clone 3TW”. The nucleotide sequence of clone 3TW isreported as SEQ ID NO:13. Nucleotides 3 to 2846 of SEQ ID NO:13 encode aTNF-R1-DD ligand protein, the amino acid sequence of which is reportedas SEQ ID NO:14. Amino acids 811 to 948 of SEQ ID NO:14 correspond toamino acids 1 to 138 of SEQ ID NO:4 (clone 3DD). Clone 3TW was depositedwith the American Type Culture Collection on Sep. 26, 1995 and given theaccession number ATCC 69904.

The sequence of a polynucleotide encoding another such protein is setforth in SEQ ID NO:5 from nucleotides 2 to 559. This polynucleotide hasbeen identified as “clone 20DD.” The amino acid sequence of theTNF-R1-DD ligand protein encoded by clone 20DD is set forth in SEQ IDNO:6. It is believed that clone 20DD is a partial cDNA clone of a longerfull length coding sequence. However, as demonstrated herein the proteinencoded by clone 20DD does bind the death domain of TNF-R (i.e., has“TNF-R1-DD ligand protein activity” as defined herein). Clone 20DD wasdeposited with the American Type Culture Collection on Oct. 13, 1994 andgiven the accession number ATCC 69704.

The protein encoded by clone 20DD is identical to amino acids 87 to 272of insulin-like growth factor binding protein-5 (“IGFBP-5”), a sequencefor which was disclosed in J. Biol. Chem. 266:10646-10653 (1991) byShimasaki et al., which is incorporated herein by reference. Thepolynucleotide and amino acid sequences of IGFBP-5 are set forth in SEQID NO:7 and SEQ ID NO:8, respectively. Based upon the sequence identitybetween clone 20DD and IGFBP-5, IGFBP-5 and certain fragments thereofwill exhibit TNF-R1-DD ligand binding activity (as defined herein).

The sequence of a polynucleotide encoding another such protein is setforth in SEQ ID NO:9 from nucleotides 2 to 931. This polynucleotide hasbeen identified as “clone 1TU” The amino acid sequence of the TNF-R1-DDligand protein encoded by clone 1TU is set forth in SEQ ID NO: 10. It isbelieved that clone 1TU is a partial cDNA clone of a longer full lengthcoding sequence. However, as demonstrated herein the protein encoded byclone 1TU does bind the death domain of TNF-R (i.e., has “TNF-R1-DDligand protein activity” as defined herein). Clone 1TU was depositedwith the American Type Culture Collection on Jun. 7, 1995 and given theaccession number ATCC 69848.

The protein encoded by clone 1TU is 310 amino acids in length. Noidentical or closely related sequences were found using BLASTN/BLASTX orFASTA searches. Therefore clone 1TU encodes a novel protein.

The sequence of a polynucleotide encoding another such protein is setforth in SEQ ID NO:11 from nucleotides 2 to 1822. This polynucleotidehas been identified as “clone 27TU” The amino acid sequence of theTNF-R1-DD ligand protein encoded by clone 27TU is set forth in SEQ IDNO:12. It is believed that clone 27TU is a partial cDNA clone of alonger full length coding sequence. However, as demonstrated herein theprotein encoded by clone 27TU does bind the death domain of TNF-R (i.e.,has “TNF-R1-DD ligand protein activity” as defined herein). Clone 27TUwas deposited with the American Type Culture Collection on Jun. 7, 1995and given the accession number ATCC 69846.

The protein encoded by clone 27TU is 607 amino acids in length. Noidentical or closely related sequences were found using BLASTN/BLASTX orFASTA searches. Therefore, clone 27TU encodes a novel protein. 27TU maybe a longer version of clone 2DD. 2DD encodes the same amino acidsequence (SEQ ID NO:2) as amino acids 198-607 encoded by 27TU (SEQ IDNO:12). The nucleotide sequences of 2DD and 27TU are also identicalwithin this region of identity.

An additional “clone 15TU” was isolated which encoded a portion of the27TU sequence (approximately amino acids 289-607 of SEQ ID NO:12). Clone15TU was deposited with the American Type Culture Collection on Jun. 7,1995 and given the accession number ATCC 69847. 15TU comprises the samenucleotide sequence as 27TU over this region of amino acids.

A full-length clone corresponding to clone 27TU was also isolated andidentified as “clone 57TU4A”. The nucleotide sequence of clone 57TU4A isreported as SEQ ID NO:15. Nucleotides 336 to 5092 of SEQ ID NO:15 encodea TNF-R1-DD ligand protein, the amino acid sequence of which is reportedas SEQ ID NO:146 Amino acids 982 to 1588 of SEQ ID NO:16 correspond toamino acids 1 to 607 of SEQ ID NO: 12 (clone 27TU). Clone 57TU4A wasdeposited with the American Type Culture Collection on Feb. 13, 1996 andgiven the accession number ATCC 69988.

A full-length clone corresponding to clone 1TU was also isolated andidentified as “clone 33-1B”. The nucleotide sequence of clone 33-1B isreported as SEQ ID NO:17. Nucleotides 14 to 2404 of SEQ ID NO:17 encodea TNF-R1-DD ligand protein the amino acid sequence of which is reportedas SEQ ID NO:18. Amino acids 488 to 797 of SEQ ID NO:18 correspond toamino acids 1 to 310 of SEQ ID NO:10 (clone 1TU). Clone 33-1B wasdeposited with the American Type Culture Collection on Aug. 13, 1996 andgiven the accession number ATCC ______

Polynucleotides hybridizing to the polynucleotides of the presentinvention under stringent conditions and highly stringent conditions arealso part of the present invention. As used herein, “highly stringentconditions” include, for example, 0.2×SSC at 65° C.; and “stringentconditions” include, for example, 4×SSC at 65° C. or 50% formamide and4×SSC at 42° C.

For the purposes of the present application, “TNF-R1-DD ligand protein”includes proteins which exhibit TNF-R1-DD ligand protein activity. Forthe purposes of the present application, a protein is defined as having“TNF-R1-DD ligand protein activity” when it binds to a protein derivedfrom the TNF-R death domain. Activity can be measured by using any assaywhich will detect binding to an TNF-R death domain protein. Examples ofsuch assays include without limitation the interaction trap assays andassays in which TNF-R death domain protein which is affixed to a surfacein a manner conducive to observing binding, including without limitationthose described in Examples 1 and 3. As used herein an “TNF-R deathdomain protein” includes the entire death domain or fragments thereof.

Fragments of the TNF-R1-DD ligand protein which are capable ofinteracting with the TNF-R death domain or which are capable ofinhibiting TNF-R death domain binding (i.e., exhibit TNF-R1-DD ligandprotein activity) are also encompassed by the present invention.Fragments of the TNF-R1-DD ligand protein may be in linear form or theymay be cyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of TNF-R1-DD ligand protein bindingsites. For example, fragments of the TNF-R1-DD ligand protein may befused through “linker” sequences to the Fc portion of an immunoglobulin.For a bivalent form of the TNF-R1-DD ligand protein, such a fusion couldbe to the Fc portion of an IgG molecule. Other immunoglobulin isotypesmay also be used to generate such fusions. For example, an TNF-R1-DDligand protein IgM fusion would generate a decavalent form of theTNF-R1-DD ligand protein of the invention.

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the TNF-R1-DD ligand protein recombinantly.Many suitable expression control sequences are known in the art. Generalmethods of expressing recombinant proteins are also known and areexemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). Asdefined herein “operably linked” means that the isolated polynucleotideof the invention and the expression control sequence are situated withina vector or cell in such a way that the TNF-R1-DD ligand protein isexpressed by a host cell which has been transformed (transfected) withthe ligated polynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the TNF-R1-DD ligand protein. Host cells include, for example, monkeyCOS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells,human epidermal A43 1 cells, human Colo205 cells, 3T3 cells, CV-1 cells,other transformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

The TNF-R1-DD ligand protein may also be produced by operably linkingthe isolated polynucleotide of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac®D kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987.),incorporated herein by reference.

Alternatively, it may be possible to produce the TNF-R1-DD ligandprotein in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the TNF-R1-DD ligandprotein is made in yeast or bacteria it may be necessary to modify theprotein produced therein, for example by phosphorylation orglycosylation of the appropriate sites, in order to obtain thefunctional TNF-R1-DD ligand protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

The TNF-R1-DD ligand protein of the invention may also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a nucleotide sequence encoding theTNF-R1-DD ligand protein.

The TNF-R1-DD ligand protein of the invention may be prepared byculturing transformed host cells under culture conditions suitable toexpress the recombinant protein. The resulting expressed protein maythen be purified from such culture (i.e., from culture medium or cellextracts) using known purification processes, such as gel filtration andion exchange chromatography. The purification of the TNF-R1-DD ligandprotein may also include an affinity column containing the TNF-R deathdomain or other TNF-R death domain protein; one or more column stepsover such affinity resins as concanavalin A-agarose, heparin-toyopearl®or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobicinteraction chromatography using such resins as phenyl ether, butylether, or propyl ether; or immunoaffinity chromatography.

Alternatively, the TNF-R1-DD ligand protein of the invention may also beexpressed in a form which will facilitate purification. For example, itmay be expressed as a fusion protein, such as those of maltose bindingprotein (MBP) or glutathione-S-transferase (GST). Kits for expressionand purification of such fusion proteins are commercially available fromNew England BioLab (Beverly, Mass.) and Pharmacia (Piscataway, N.J.),respectively. The TNF-R ligand protein can also be tagged with anepitope and subsequently purified by using a specific antibody directedto such epitope. One such epitope (“Flag”) is commercially availablefrom Kodak (New Haven. Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media. e.g.silica gel having pendant methyl or other aliphatic groups, can beemployed to further purity, the TNF-R1-DD ligand protein. Some or all ofthe foregoing purification steps in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinantprotein. The TNF-R1-DD ligand protein thus purified is substantiallyfree of other mammalian proteins and is defined in accordance with thepresent invention as an “isolated TNF-R1-DD ligand protein.”

TNF-R1-DD ligand proteins may also be produced by known conventionalchemical synthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with TNF-R1-DD ligand proteins may possess biologicalproperties in common therewith, including TNF-R1-DD ligand proteinactivity. Thus, they may be employed as biologically active orimmunological substitutes for natural, purified TNF-R1-DD ligandproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The TNF-R1-DD ligand proteins provided herein also include proteinscharacterized by amino acid sequences similar to those of purifiedTNF-R1-DD ligand proteins but into which modification are naturallyprovided or deliberately engineered. For example, modifications in thepeptide or DNA sequences can be made by those skilled in the art usingknown techniques. Modifications of interest in the TNF-R1-DD ligandprotein sequences may include the replacement, insertion or deletion ofa selected amino acid residue in the coding sequence. For example, oneor more of the cysteine residues may be deleted or replaced with anotheramino acid to alter the conformation of the molecule. Mutagenictechniques for such replacement, insertion or deletion are well known tothose skilled in the art (see, e.g., U.S. Pat. No. 4,518,584).

Other fragments and derivatives of the sequences of TNF-R1-DD ligandproteins which would be expected to retain TNF-R1-DD ligand proteinactivity in whole or in part and may thus be useful for screening orother immunological methodologies may also be easily made by thoseskilled in the art given the disclosures herein. Such modifications arebelieved to be encompassed by the present invention.

TNF-R1-DD ligand protein of the invention may also be used to screen foragents which are capable of inhibiting or blocking binding of anTNF-R1-DD ligand protein to the death domain of TNF-R, and thus may actas inhibitors of TNF-R death domain binding and/or TNF activity. Bindingassays using a desired binding protein, immobilized or not, are wellknown in-the art and may be used for this purpose using the TNF-R1-DDligand protein of the invention. Examples 1 and 3 describe examples ofsuch assays. Appropriate screening assays may be cell-based orcell-free. Alternatively, purified protein based screening assays may beused to identify such agents. For example, TNF-R1-DD ligand protein maybe immobilized in purified form on a carrier and binding to purifiedTNF-R death domain may be measured in the presence and in the absence ofpotential inhibiting agents. A suitable binding assay may alternativelyemploy purified TNF-R death domain immobilized on a carrier, with asoluble form of a TNF-R1-DD ligand protein of the invention. AnyTNF-R1-DD ligand protein may be used in the screening assays describedabove.

In such a screening assay, a first binding mixture is formed bycombining TNF-R death domain protein and TNF-R1-DD ligand protein, andthe amount of binding in the first binding mixture (B_(o)) is measured.A second binding mixture is also formed by combining TNF-R death domainprotein, TNF-R1-DD ligand protein, and the compound or agent to bescreened, and the amount of binding in the second binding mixture (B) ismeasured. The amounts of binding in the first and second bindingmixtures are compared, for example, by performing a B/B_(o) calculation.A compound or agent is considered to be capable of inhibiting TNF-Rdeath domain binding if a decrease in binding in the second bindingmixture as compared to the first binding mixture is observed. Theformulation and optimization of binding mixtures is within the level ofskill in the art. Such binding mixtures may also contain buffers andsalts necessary to enhance or to optimize binding, and additionalcontrol assays may be included in the screening assay of the invention.

Alternatively, appropriate screening assays may be cell based. Forexample, the binding or interaction between an TNF-R ligand protein andthe TNF-R death domain can be measured in yeast as described below inExamples 1 and 3.

Compounds found to reduce, preferably by at least about 10%, morepreferably greater than about 50% or more, the binding activity ofTNR-R1-DD ligand protein to TNF-R death domain may thus be identifiedand then secondarily screened in other binding assays, including in vivoassays. By these means compounds having inhibitory activity for TNF-Rdeath domain binding which may be suitable as anti-inflammatory agentsmay be identified.

Isolated TNF-R1-DD ligand protein may be useful in treating, preventingor ameliorating inflammatory conditions and other conditions, such ascachexia, autoimmune disease, graft versus host reaction, osteoporosis,colitis, myelogenous leukemia, diabetes, wasting, and atherosclerosis.Isolated TNF-R1-DD ligand protein may be used itself as an inhibitor ofTNF-R death domain binding or to design inhibitors of TNF-R death domainbinding. Inhibitors of binding of TNF-R1-DD ligand protein to the TNF-Rdeath domain (“TNF-R intracellular binding inhibitors”) are also usefulfor treating such conditions.

The present invention encompasses both pharmaceutical compositions andtherapeutic methods of treatment or use which employ isolated TNF-R1-DDligand protein and/or binding inhibitors of TNF-R intracellular binding.

Isolated TNF-R1-DD ligand protein or binding inhibitors (from whateversource derived, including without limitation from recombinant andnon-recombinant cell lines) may be used in a pharmaceutical compositionwhen combined with a pharmaceutically acceptable carrier. Such acomposition may also contain (in addition to TNF-R1-DD ligand protein orbinding inhibitor and a carrier) diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s). The characteristics of the carrier will dependon the route of administration. The pharmaceutical composition of theinvention may also contain cytokines, lymphokines, or otherhematopoietic factors such as M-CSF, GM-CSF, TNF, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, G-CSF, Meg-CSF, stem cell factor, anderythropoietin. The pharmaceutical composition may further contain otheranti-inflammatory agents. Such additional factors and/or agents may beincluded in the pharmaceutical composition to produce a synergisticeffect with isolated TNF-R1-DD ligand protein or binding inhibitor, orto minimize side effects caused by the isolated TNF-R1-DD ligand proteinor binding inhibitor. Conversely, isolated TNF-R1-DD ligand protein orbinding inhibitor may be included in formulations of the particularcytokine lymphokine other hematopoietic factor thrombolytic oranti-thrombotic factor or anti-inflammatory agent to minimize sideeffects of the cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of aliposome in which isolated TNF-R1-DD ligand protein or binding inhibitoris combined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithin, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. No. 4,235,871; U.S.Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323, all of which are incorporated herein by reference.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of an inflammatoryresponse or condition, or an increase in rate of treatment, healing,prevention or amelioration of such conditions. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of isolated TNF-R1-DD ligand protein orbinding inhibitor is administered to a mammal having a condition to betreated. Isolated TNF-R1-DD ligand protein or binding inhibitor may beadministered in accordance with the method of the invention either aloneor in combination with other therapies such as treatments employingcytokines, lymphokines or other hematopoietic factors. Whenco-administered with one or more cytokines, lymphokines or otherhematopoietic factors, isolated TNF-R1-DD ligand protein or bindinginhibitor may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering isolated TNF-R1-DD ligand protein or bindinginhibitor in combination with cytokine(s), lymphokine(s), otherhematopoietic factor(s), thrombolytic or anti-thrombotic factors.

Administration of isolated TNF-R1-DD ligand protein or binding inhibitorused in the pharmaceutical composition or to practice the method of thepresent invention can be carried out in a variety of conventional ways,such as oral ingestion, inhalation, or cutaneous, subcutaneous, orintravenous injection. Intravenous administration to the patient ispreferred.

When a therapeutically effective amount of isolated TNF-R1-DD ligandprotein or binding inhibitor is administered orally, isolated TNF-R1-DDligand protein or binding inhibitor will be in the form of a tablet,capsule, powder, solution or elixir. When administered in tablet form,the pharmaceutical composition of the invention may additionally containa solid carrier such as a gelatin or an adjuvant. The tablet, capsule,and powder contain from about 5 to 95% isolated TNF-R1-DD ligand proteinor binding inhibitor, and preferably from about 25 to 90% isolatedTNF-R1-DD ligand protein or binding inhibitor. When administered inliquid form, a liquid carrier such as water, petroleum, oils of animalor plant origin such as peanut oil, mineral oil, soybean oil, or sesameoil, or synthetic oils may be added. The liquid form of thepharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5 to 90% by weight of isolated TNF-R1-DD ligand protein orbinding inhibitor, and preferably from about 1 to 50% isolated TNR-R1-DDligand protein or binding inhibitor.

When a therapeutically effective amount of isolated TNF-R1-DD ligandprotein or binding inhibitor is administered by intravenous, cutaneousor subcutaneous injection, isolated TNF-R1-DD ligand protein or bindinginhibitor will be in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such parenterally acceptableprotein solutions having due regard to pH, isotonicity stability and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to isolated TNF-R1-DD ligand protein or bindinginhibitor, an isotonic vehicle such as Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, Lactated Ringer's Injection, or other vehicle as known in theart. The pharmaceutical composition of the present invention may alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The amount of isolated TNF-R1-DD ligand protein or binding inhibitor inthe pharmaceutical composition of the present invention will depend uponthe nature and severity of the condition being treated, and on thenature of prior treatments which the patient has undergone. Ultimately,the attending physician will decide the amount of isolated TNF-R1-DDligand protein or binding inhibitor with which to treat each individualpatient. Initially, the attending physician will administer low doses ofisolated TNF-R1-DD ligand protein or binding inhibitor and observe thepatient's response. Larger doses of isolated TNF-R1-DD ligand protein orbinding inhibitor may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.1 μg to about 100 mg of isolated TNF-R1-DD ligandprotein or binding inhibitor per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the isolated TNF-R1-DD ligand protein orbinding inhibitor will be in the range of 12 to 24 hours of continuousintravenous administration. Ultimately the attending physician willdecide on the appropriate duration of intravenous therapy using thepharmaceutical composition of the present invention.

Isolated TNF-R1-DD ligand protein of the invention may also be used toimmunize animals to obtain polyclonal and monoclonal antibodies whichspecifically react with the TNF-R1-DD ligand protein and which mayinhibit TNF-R death domain binding. Such antibodies may be obtainedusing either the entire TNF-R1-DD ligand protein or fragments ofTNF-R1-DD ligand protein as an immunogen. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus andare conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Methods for synthesizing such peptides are known in the art, forexample, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154(1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

Monoclonal antibodies binding to TNF-R1-DD ligand protein or to complexcarbohydrate moieties characteristic of the TNF-R1-DD ligandglycoprotein may be useful diagnostic agents for the immunodetection ofTNF-R ligand protein.

Neutralizing monoclonal antibodies binding to TNF-R1-DD ligand proteinor to complex carbohydrates characteristic of TNF-R1-DD ligandglycoprotein may also be useful therapeutics for both inflammatoryconditions and also in the treatment of some forms of cancer whereabnormal expression of TNF-R1-DD ligand protein is involved. Theseneutralizing monoclonal antibodies are capable of blocking the signalingfunction of the TNF-R1-DD ligand protein. By blocking the binding ofTNF-R1-DD ligand protein, certain biological responses to TNF are eitherabolished or markedly reduced. In the case of cancerous cells orleukemic cells, neutralizing monoclonal antibodies against TNF-R1-DDligand protein may be useful in detecting and preventing the metastaticspread of the cancerous cells, which may be mediated by the TNF-R1-DDligand protein.

Due to the similarity of their sequences to the insulin growth factorbinding protein (“IGFBP-5”) and fragments thereof which bind to theTNF-R death domain are proteins having TNF-R1-DD ligand protein activityas defined herein. As a result, they are also useful in pharmaceuticalcompositions, for treating inflammatory conditions and for inhibitingTNF-R death domain binding as described above for TNF-R1-DD ligandproteins generally.

EXAMPLE 1 Cloning of TNF-R Death Domain Ligand Protein EncodingPolynucleotide

A yeast genetic selection method, the “interaction trap” [Gyuris et al.Cell 75:791-803, 1993, which is incorporated herein by reference], wasused to screen WI38 cell cDNA libraries (preparation, see below) forproteins that interact with the death domain of the P55 type 1 TNFreceptor (TNF-R1-DD). A polynucleotide encoding amino acids 326 to 413of the P55 type TNF receptor. TNF-R1-DD with obtained via the polymerasechain reaction (PCR) using a grafting method. This TNF-R1-DD DNA wasthen cloned into pEG202 by BamHI and SalI sites, generating the baitplasmid, pEG202-TNF-R1-DD. This plasmid contains the HIS3 selectablemarker, and expression of the bait, the LexA-TNF-R1-DD fusion protein,is from the strong constitutive ADH1 promoter. To create the reporterstrain carrying the bait protein, yeast strain EGY48, containing thereporter sequence LexAop-Leu2 in place of the chromosomal LEU2, wastransformed with pEG202-TNF-R1-DD and pSH18-34 (Ura+), which carriesanother reporter sequence, LexAop-lacZ. For screening cDNAs encodingproteins that interact with TNF-R1-DD, the expression vector pJG4-5(TRP1), containing the WI38 cell cDNA library (see below for the cDNAlibrary construction), was transformed into the above strain(EGY48/pEG202-TNF-R1-DD/pSH18-34) according to the method described byGietz et al., Nucleic Acids Res., 20:1425 (1992).

cDNA Library Construction:

WI38 cell cDNA library: Double stranded cDNA was prepared from 3 ug ofWI38 mRNA using reagents provided by the Superscript Choice System(Gibco/BRL, Gaithersberg, Md.) with the following substitutions: thefirst strand synthesis was primed using an oligo dT/XhoI primer/linker,and the dNTP mix was substituted with a mix containing methyl dCTP(Stratagene, LaJolla, Calif.). The cDNA was modified at both ends byaddition of an EcoRI/NotI/SalI adapter linker and subsequently digestedwith XhoI. This produced cDNA molecules possessing an EcoRI/NotI/SalIoverhang at the 5′ end of the gene and an XhoI overhang at the 3′ end.These fragments were then ligated into the yeast expression/fusionvector pJG4-5 (Gyuris et al., Cell, 75, 791-803, 1993), which containsat its amino terminus, the influenza virus HA1 epitope tag, the B42acidic transcription activation domain, and the SV40 nuclearlocalization signal, all under the control of the galactose-dependentGAL1 promoter. The resulting plasmids were then electroporated intoDH10B cells (Gibco/BRL). A total of 7.1×10⁶ colonies were plated on LBplates containing 100 ug/ml of ampicillin. These E. coli were scraped,pooled, and a large scale plasmid prep was performed using the WizardMaxi Prep kit (Promega, Madison, Wis.), yielding 3.2 mg of supercoiledplasmid DNA.

WI38 Cell cDNA Screening Results:

1×10⁶ transformants were obtained on glucose Ura His Trp plates. Thesetransformants were pooled and resuspended in a solution of 65% glycerol,10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂ and stored at −80° C. in 1 mLaliquots. For screening purposes, aliquots of these were diluted 10-foldinto Ura His Trp CM dropout gal/raff medium (containing 2% galactose, 1%raffinose), which induces the expresssion of the library encodedproteins, and incubated at 30° C. for 4 hours. 12×10⁶ colony formingunits (CFUs) were then plated on standard 10 cm galactose X-Gal Ura HisTrp Leu plates at a density of 2×10⁵ CFU/plate. After three days at 30°C., about 1,000 colonies were formed (Leu⁺) and of those, sixty-fourcolonies were LacZ⁺. In order to test if the Leu⁺/LacZ⁺ phenotype wasdue to the library-encoded protein, the galactose dependency of thephenotype was tested. Expression of the library-encoded proteins wasturned off by growth on glucose Ura His Trp master plates and thenretested for galactose-dependency on glucose Ura His Trp Leu, galactoseUra His Trp Leu, glucose X-Gal Ura His Trp; and galactose X-Gal Ura HisTrp plates. Of these, 32 colonies showed galactose-dependent growth onLeu plates and galactose-dependent blue color on X-Gal-containing medium(LacZ⁺ phenotype). Total yeast DNA was prepared from these coloniesaccording to the method described previously (Hoffman and Winston,1987). In order to analyze the cDNA sequences, PCR reactions wereperformed using the above yeast DNA as a template and oligo primersspecific for the vector pJG4-5, flanking the cDNA insertion point. PCRproducts were purified (Qiagen PCR purification kit), subjected torestriction digest with the enzyme HaeIII, run on 1.8% agarose gels, andthe restriction patterns compared. Similar and identical restrictionpatterns were grouped and representatives of each group were sequencedand compared to Genbank and other databases to identify any sequencehomologies.

One clone of unique sequence (“2DD”) and three clones with identicalsequence (“3DD”) were isolated and showed no significant sequencehomologies compared to Genbank and other databases. Additionally, fourother clones (“20DD”) with identical sequence to a portion of humaninsulin-like growth factor binding protein-5 (Shunichi Shimasaki et al.,J. Biol. Chem. 266:10646-10653 (1991)) were isolated. The clones “2DD,”“3DD” and “20DD” were chosen for further analysis. Library vector pJG4-5containing these clones sequence were rescued from yeast by transformingthe total yeast DNAs into the E. coli strain KC8 and selecting forgrowth on Trp-ampicillin plates. These putative TNFR1 interactingproteins were then tested further for specificity of interaction withthe TNF-R1-DD by the reintroduction of JG4-5 clone into EGY48derivatives containing a panel of different baits, including bicoid, thecytoplasmic domain of the IL-1 receptor, and TNF-R1-DD. The above cloneswere found to interact only with the TNF-R1-DD. The interaction betweenthese clones and TNF-R1-DD was thus judged to be specific.

U937 cDNA Screening Results:

A U937 cDNA library was also constructed and screened as describedabove. 1,020 Leu+ colonies were found and of those, 326 colonies werealso LacZ+. 62 colonies of these Leu+/LacZ+ colonies showed agalactose-dependent phenotype. One of these clones, 1TU, encodes a novelsequence. Interestingly, two clones, 15TU and 27TU, encode related oridentical sequences, except that 27TU contains about 864 additionalnucleotides (or about 288 amino acids) at the 5′ end. 15/27TU alsoencode a novel sequence.

EXAMPLE 2 Expression of the TNF-R1-DD Ligand Protein

cDNAs encoding TNF-R intracellular ligand proteins were released fromthe pJG4-5 vector with the appropriate restriction enzymes. For example,EcoRI and XhoI or NotI and XhoI were used to release cDNA from clone 2DDand clone 20DD. Where the restriction sites were also present in theinternal sequence of the cDNA, PCR was performed to obtain the cDNA. Forexample, the cDNA fragment encoding “clone 3DD” was obtained through PCRdue to the presence of an internal XhoI site. These cDNAs were thencloned into various expression vectors. These included pGEX (Pharmacia)or pMAL (New England Biolabs) for expression as a GST(Glutathione-S-transferase) or MBP (maltose binding protein) fusionprotein in E. coli, a pED-based vector for mammalian expression and pVLor pBlueBacHis (Invitrogen) for baculovirus/insect expression. For theimmunodetection of TNF-R intracellular ligand expression in mammaliancells, an epitope sequence, “Flag.” was inserted into the translationalstart site of the pED vector, generating the pED-Flag vector. cDNAs werethen inserted into the pED-Flag vector. Thus, the expression of cDNAfrom pED-Flag yields a protein with an amino terminal Met, followed bythe “Flag” sequence, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. StandardDEAE-Dextran or lipofectamine methods were used to transfect COS or CHOdukx cells. Immunodetection of Flag-tagged proteins was achieved usingthe M2 antibody (Kodak). Moreover, an immunoaffinity column using the M2antibody, followed by elution with the “Flag” peptide, can be used forthe rapid purification of the flag-tagged protein. Similarly, affinitypurification of GST-, MBP- or His-tagged fusion proteins can beperformed using glutathione, amylose, or nickel columns. Detailedpurification protocols are provided by the manufacturers. For manyfusion proteins, the TNF-R intracellular ligand can be released by theaction of thrombin, factor Xa, or enterokinase cleavage. In the casewhere highly purified material is required, standard purificationprocedures, such as ion-exchange, hydrophobic, and gel filtrationchromatography will be applied in addition to the affinity purificationstep.

FIGS. 1 and 2 depict autoradiographs demonstrating the expression ofTNF-R1-DD ligand proteins in yeast and mammalian cells. FIG. 1 shows theresults of expression of isloated clones of the present invention inyeast. EGY48 was transformed with pJG4-5 containing clone 2DD, 3DD or20DD. Cells were then grown overnight in the galactose/raffinose medium.Cell lysates were prepared and subject to 4-20% SDS gel electrophoresis,followed by Western blot analysis using anti-HA antibody (12CA5,Boehringer Mannheim, Indianapolis, Ind.). FIG. 2 shows the results ofexpression of Flag-2DD and Flag-20DD in COS cells. COS cells weretransfected with either pED-Flag (Vector control), Flag-2DD or Flag-20DDplasmid by the lipofectamine method. Thirty μg of each cell lysate wereprepared and subjected to 4-20% SDS gel electrophoresis, followed byWestern blot analysis using M2 antibody (Kodak). The bands in theFlag-2DD and Flag-20DD lanes indicate significant expression of therespective TNF-R1-DD ligand proteins.

EXAMPLE 3 Assays of TNF-R Death Domain Binding

Two different methods were used to assay for TNF-R1-DD ligand proteinactivity. The first assay measures binding in the yeast strain in“interaction trap,” the system used here to screen for TNF-R1-DDinteracting proteins. In this system, the expression of reporter genesfrom both LexAop-Leu2 and LexAop-LacZ relies on the interaction betweenthe bait protein, in this case TNF-R1DD, and the prey, the TNF-Rintracellular ligand. Thus, one can measure the strength of theinteraction by the level of Leu2 or LacZ expression. The most simplemethod is to measure the activity of the LacZ encoded protein,β-galactosidase. This activity can be judged by the degree of bluenesson the X-Gal containing medium or filter. For the quantitativemeasurement of β-galactosidase activity, standard assays can be found in“Methods in Yeast Genetics” Cold Spring Harbor, N.Y., 1990 (by Rose, M.D., Winston, F., and Hieter, P.).

The second assay for measuring binding is a cell-free system. An exampleof a typical assay is described below. Purified GST-TNF-R1-DD fusionprotein (2 ug) was mixed with amylose resins bound with a GST-TNF-R1-DDintracellular ligand for 2 hour at 4° C. The mixture was thencentrifuged to separate bound (remained with the beads) and unbound(remained in the supernatant) GST-TNF-R1-DD. After extensive washing,the bound GST-TNF-R1-DD was eluted with maltose and detected by Westernblot analysis using a GST antibody. The TNF-R1-DD or the intracellularligand can also be immobilized on other solid supports, such as onplates or fluorobeads. The binding can then be measured using ELISA orSPA (scintillation proximity assay).

EXAMPLE 4 Characterization of TNF-R Death Domain Ligand Protein

Mapping the Interaction Site in TNF-R1

Many of the key amino acids for TNF-R signaling have been determined bysite-directed mutagenesis (Tataglia et at., Cell 74:845-853 (1993).These amino acids are conserved between TNF-R and the Fas antigen, whichis required for mediating cytotoxicity and other cellular responses. Inorder to test if the TNF-R intracellular proteins interact with theseresidues, the following mutations were constructed: F345A (substitutionof phe at amino acid 345 to Ala), R347A, L351A, F345A/R347A/L351A,E369A, W378A and I408A. The ability of the mutant protein to interactwith the intracellular ligand in the “interaction trap” system wastested.

Effect on the TNF-Mediated Response

The effect of the TNF-R intracellular ligands on the TNF-mediatedresponse can be evaluated in cells overexpressing the ligands. A numberof TNF-mediated responses, including transient or prolonged responses,can be measured. For example, TNF-induced kinase activity toward eitherMBP (myelin basic protein) or the N-terminus (amino acids 1-79) of c-juncan be measured in COS cells or CHO cells either transiently or stablyoverexpressing clone 2DD, 3DD or clone 20DD. The significance of theseligand proteins in TNF-mediated cytotoxicity and other cellularresponses can be measured in L929 or U937 overexpressing cells.Alternatively, other functional assays, such as the induction of geneexpression or PGE₂ production after prolonged incubation with TNF, canalso be used to measure the TNF mediated response. Conversely, thesignificance of the TNF-R1-DD ligand proteins in TNF signaling can beestablished by lowering or eliminating the expression of the ligands.These experiments can be performed using antisense expression ortransgenic mice.

Enzymatic or Functional Assays

The signal transduction events initiated by TNF binding to its receptorare still largely unknown. However, one major result of TNF binding isthe stimulation of cellular serine/threonine kinase activity. Inaddition, TNF has been shown to stimulate the activity of PC-PLC, PLA₂,and sphingomyelinase. Therefore, some of the TNF-R1-DD ligand proteinsmay possess intrinsic enzymatic activity that is responsible for theseactivities. Therefore, enzymatic assays can be performed to test thispossibility, particularly with those clones that encode proteins withsequence homology to known enzymes. In addition to enzymatic activity,based on the sequence homology to proteins with known function, otherfunctional assays can also be measured.

EXAMPLE 5 Isolation of Full Length Clones

In many cases, cDNAs obtained from the interaction trap method eachencode only a portion of the full length protein. For example, based onidentity and sequence and the lack of the initiating methionine codon,clones 2DD, 3DD and 20DD apparently do not encode full length proteins.Therefore, it is desirable to isolate full length clones. The cDNAsobtained from the screening, such as clone 2DD, are used as probes, andthe cDNA libraries described herein, or alternatively phage cDNAlibraries, are screened to obtain full length clones in accordance withknown methods (see for example, “Molecular Cloning, A LaboratoryManual”, by Sambrook et al., 1989 Cold Spring Harbor).

EXAMPLE 6 Antibodies Specific for TNF-R Intracellular Ligand Protein

Antibodies specific for TNF-R intracellular ligand proteins can beproduced using purified recombinant protein, as described in Example 2,as antigen. Both polyclonal and monoclonal antibodies will be producedusing standard techniques, such as those described in “Antibodies, aLaboratory Manual” by Ed Harlow and David Lane (1988), Cold SpringHarbor Laboratory.

EXAMPLE 7 Characterization of Clones 1TU and 15/27TU

Specificity of Interaction

The specificity of clones 1TU, 15TU and 27TU was tested using a panel ofbaits. The ability of these clones to bind the TNF-R death domain wascompared to their binding to the intracellular domain of the secondTNF-R (TNF-R p75_(IC)), the entire intracellular domain of TNF-R (TNF-Rp55_(IC)), the death domain of the fas antigen (which shares 28%identity with TNF-R-DD) (Fas_(DD)), the Drosoplhila transcription factorbicoid, and a region of the IL-1 receptor known to be critical forsignalling (IL-1R₄₇₇₋₅₂₇). As shown in Table 1, none of these clonesinteracted with TNF-R p75_(IC) or Fas_(DD), and only 1TU interacted withbicoid. In contrast, both 1TU and 15TU bound the cytoplasmic domain ofthe p55 TNF-R, as well as residues 477-527 of the IL-1R. 27TU interactedrelatively weakly with these sequences. TABLE 1 TNF-R TNF-R IL-IR cloneTNF-R_(DD) p75_(IC) p55_(IC) Fas_(DD) bicoid (477-527)  1TU +++ − +++ −++ +++ 15TU +++ ± +++ − − ++ 27TU +++ − + − − +Interaction with Amino Acids Critical for Signalling

The ability of each clone to interact with four single-site mutations inthe TNF-R death domain (each known to abolish signalling) was measured.As shown in Table 2, each of the clones interacted less strongly withthe death domain mutants than with the wild type death domain,suggesting that these clones may bind critical residues in vivo. TABLE 2clone TNF-R_(DD) F345A L351A W378A I408A  1TU +++ + ++ ++ + 15TU +++ + +++ ++ 27TU +++ + + ± ++Expression of 1TU, 15TU and 27TU

FIG. 3 depicts an autoradiograph demonstrating the expression of clones1TU, 15TU and 27TU in yeast (A) and COS cells (B).

In (A): EGY48 was transformed with pJG4-5 containing clones 1TU, 15TU or27TU. Cells were then grown overnight in galactose/raffinose medium.Cell lysates were prepared and subjected to 4-20% SDS gelelectropho-esis. followed by Western blot analysis using anti-HAantibody (12CA5, Boehringer Mannheim).

In (B): COS cells were transfected with pED-Flag containing clones 1TU,15TU and 27TU. Cell lysates were prepared and analyzed by Western blotusing anti-Flag antibody (M2, Kodak).

Specific Binding of 1TU and 27TU to TNF-R1-DD

The interaction of 1TU and 27TU with TNF-R1-DD was tested using purifiedbacterially expressed fusion proteins. As shown in FIG. 4, MBP fusionproteins containing 1TU or 27TU bound only to TNF-R1-DD expressed as aGST fusion protein, but not to GST protein alone. In the controlexperiment, MBP protein did not bind either GST or GST/TNF-R1-DD. Theseresults indicate that 1TU and 27TU bound specifically to the TNF-R1deathdomain in vitro, confirming the data obtained in the interaction trap.

15TU and 27TU Activation of JNK Activity

The jun N-terminal kinase (JNK) is normally activated within 15 min ofTNF treatment-in COS cells. 15TU and 27TU were cotransfected with anepitope tagged version of JNK, HA-JNK, in duplicate. After TNFtreatment, JNK was immunoprecipitated with anti-HA antibody and JNKactivity was measured in immunoprecipitation kinase assays, usingGST-c-jun (amino acids 1-79) as substrate). Reactions wereelectrophoresed on SDS-PAGE. As shown in FIG. 5, transfection of 15TUand 27TU, but not vector alone, into COS cells activated JNK even in theabsence of TNF, suggesting that these clones are involved in signaltransduction of TNF and the pathway leading to JNK activation in vivo.

EXAMPLE 8 Isolation, Expression and Assay of Clone 3TW

Clone 3TW was isolated from the W138 cDNA library using clone 3DD as aporbe. Clone 3TW was expressed. FIG. 6 is an autoradiograph whichdemonstrates expression of 3TW (indicated by arrow).

An antisense oligonucleotide was derived from the sequence of clone 3TW.The antisense oligonucleotide was assayed to determine its ability toinhibit TNF-induced cPLA₂ phosphorylation. FIG. 7 depicts the results ofthat experiment. Activity of the anitsense oligonucleotide (3TWAS) wascompared with the full-length clone (3TWFL), Flag-3TW full length(3TWFLflag) and pED-flag vector (pEDflag). The antisense oligonucleotideinhibited phosphorylation.

1-46. (canceled)
 47. A method of preventing or ameliorating aninflammatory disorder by administering to a mammal an antibody whichspecifically binds to a protein with the amino acid sequence of SEQ NO:2or a fragment of said antibody.
 48. The method of claim 47, wherein saidantibody is a neutralizing antibody.
 49. The method of claim 47, whereinsaid antibody blocks binding of TNF-R1-DD to a polypeptide TNF-R1-DDligand protein with the amino acid sequence of SEQ ID NO:2.
 50. Themethod of claim 47, wherein said antibody is a polyclonal antibody. 51.The method of claim 47, wherein said antibody is a monoclonal antibody.52. The method of claim 47, wherein said antibody specifically reactswith a protein comprising the amino acid sequence of SEQ ID NO:2. 53.The method of claim 47, wherein said antibody specifically reacts with aprotein comprising the amino acid sequence of SEQ ID NO:12.
 54. Themethod of claim 47, wherein said antibody specifically reacts with aprotein comprising the amino acid sequence of SEQ ID NO:16.
 55. Themethod of claim 47, wherein said disorder is cachexia, autoimmunedisease, graft-versus-host reaction, osteoporosis, colitis, myelogenousleukemia, diabetes, wasting, or atherosclerosis.
 56. A method ofpreventing or ameliorating an inflammatory disorder by administering toa mammal a neutralizing antibody that binds specifically to a proteinwith the amino acid sequence of SEQ ID NO:2, wherein said neutralizingantibody blocks binding of TNF-R1-DD to a TNF-R1-DD ligand protein withthe amino acid sequence of SEQ ID NO:2, or a fragment of saidneutralizing antibody.
 57. The method of claim 56, wherein said antibodyis an isolated antibody.
 58. A method of preventing or ameliorating aninflammatory disorder by administering to a mammal an antibody thatspecifically binds to a protein with the amino acid sequence of SEQ IDNO:2, wherein said antibody is obtained using as an immunogen TNF-R1-DDligand protein with the amino acid sequence of SEQ ID NO:2.
 59. Themethod of claim 58, wherein said antibody is obtained using as animmunogen a protein with tyrosine residues replaced with sulfatedtyrosine residues.
 60. The method of claim 58, wherein said immunogen isconjugated to a hapten.
 61. The method of claim 58, wherein said haptenis keyhole limpet hemocyanin (KLH).